Cyclases play important roles in the transduction of extracellular signals via their synthesis of “secondary messengers” such as adenosine 3′, 5′-cyclic phosphate (cyclic adenosine monophosphate, cAMP) and guanosine 3′, 5′-cyclic phosphate (cyclic guanosine monophosphate, cGMP). Cell surface receptors mediate the transduction of an extracellular signal, such as the binding of a ligand to a receptor, into a signal that is transmitted internally within the cell. The internal signal is carried by secondary messengers, which typically are produced in response to the binding of an external signal. The secondary messengers in turn activate particular proteins and other regulators within the cell which have the potential to regulate expression of specific genes or to alter a metabolic process.
Cyclic AMP and cGMP play important roles in the regulation of a multitude of cellular activities. For example, cAM responds to cellular signals through a specific protein kinase (cAMP-dependent protein kinase or protein kinase A) to phosphorylate target molecules, e.g., other protein kinases or proteins involved in transport or cellular morphology. Through stimulation of the kinase, intracellular cAMP mediates many of the effects of hormones in the regulation of cellular metabolism and cell growth. Cyclic GMP also acts as an intracellular messenger, for example, by activating cGMP-dependent kinases and regulating cGMP sensitive ion channels. The role of cGMP as a secondary messenger has been well established in vascular smooth muscle relaxation and retinal phototransduction.
Adenylate Cyclase
The synthesis of cAMP from adenosine triphosphate (ATP) is catalyzed by adenylate cyclase (also referred to as adenylyl cyclase and adenyl cyclase). In mammalian cells, adenylate cyclase is usually an integral membrane protein. Adenylate cyclase activity may be affected by a factor/receptor binding event transmitted through an associated G protein. Interaction of several different external factors with their distinct receptors causes alterations in cAMP intracellular concentration (Broach et al., U.S. Pat. No. 6,001,553). Different receptors are associated with their own particular G-protein intermediary, which itself is associated with adenylate cyclase.
At least nine distinct isoenzymes of mammalian adenylate cyclase have been identified and are designated as adenylate cyclases types 1–9 (Antoni et al., U.S. Pat. No. 6,090,612). These adenylate cyclases have a general structure consisting of 12 transmembrane helices and two cytoplasmic, catalytic domains (Hurley, 1998, Curr. Opin. Struct. Biol. 8:770–77). Some of these enzymes have been analyzed functionally and appear to confer unique signal processing capacities to cells (Taussig et al., 1995, J. Biol. Chem. 270:1–4).
In addition to functional diversity, adenylate cyclase isozymes have distinct tissue distribution profiles (Iyengar, U.S. Pat. No. 6,034,071). Localization studies using mRNA probes have been used to determine tissue distribution of the various adenylate cyclases (Pieroni et al., 1993, Curr. Opin. Neurobiol. 3: 345–351). Adenylate cyclase type 1 (AC 1) appears to be present only in neuronal tissue, whereas AC 2 has been found in brain and lung. AC 3 has been localized in olfactory neurons as well as other neuronal and non-neuronal tissues (Glatt and Snyder, 1993, Nature 361: 536–538; Xia et al., 1992, Neurosci. Lett. 144: 169–173). AC 4 appears to be present at very low levels in brain, and throughout most tissues. AC 5 and AC 6 have also been found to be widely distributed (Premont et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 9809–9813; Krupinski et al., 1992, J. Biol. Chem. 267: 24858–24862) although AC 6 seems to be of very low abundance in the brain. AC 5 is particularly abundant in the heart and in some regions of the brain. AC 7 appears to be widely distributed, but may be scarce in the brain. AC 8, like AC 1, seems to be abundant in the brain. Within the brain, the distributions of AC 1, AC 2, AC 3, AC 5 and AC 8 show distinct regional patterns. Collectively, these observations indicate that the particular adenylate cyclase isotype profile of a cell is fundamentally important with respect to cellular function.
Association with Disease
The wide variety of cyclases that have been identified thus far, together with their differing tissue distributions, demonstrate the importance of particular cellular cyclase profiles and their roles in signal transduction with respect to cellular function. Alterations in the levels of cyclic nucleotide intracellular (or secondary) messenger levels that result from alterations in particular cyclase activities have been implicated in a wide range of conditions and diseases, including cardiovascular disease, diseases of the central nervous system, intestinal conditions, retinal diseases, and shock. For instance, the high distribution of adenylate cyclases in brain has been correlated with a likely role for adenylate cyclases in neurological disorders such as Alzheimer's disease and Parkinson's disease. The high distribution of adenylate cyclase in the heart is likely to contribute to cardiovascular diseases, such as angina and hypertension.
Regulation of cyclases, therefore, has important implications for treatment of many conditions and diseases. Particular beneficial cellular responses may be elicited by blocking or stimulating the activity of particular cyclases. The many cyclases known to date and their wide distribution, coupled with their specific responses to a large number of extracellular signaling molecules, indicates that there are likely to be many more cyclases to be identified. Thus, there is a need in the art for identifying new cyclases and methods of regulating cyclase activity to provide therapeutic effects.